Oligoribonucleic acids (oligo-RNAs) are useful as RNA probes for gene analysis, RNA pharmaceutical materials (antisense RNA, ribozymes, RNA for RNAi-mediated control of gene expression), artificial enzymes, and aptamers. A solid synthesis method of preparing oligo-RNAs was established in the late 1980's. In the first report of the method, phosphoramidite compounds with tert-butyldimethylsilyl (TBDMS) or triisopropylsilyl (TIPS) as a 2′-hydroxyl protecting group were used (N. A. Usman et al., Journal of the American Chemical Society, Vol. 109, 7845 (1987)).
The chemical synthesis of oligo-RNAs presents many more problems than the chemical synthesis of oligodeoxyribonucleic acids (oligo-DNAs) made up of deoxyribonucleotides only.
For example, the use of the TBDMS group as a 2′-hydroxyl-protecting group may cause a side reaction in which the TBDMS group protecting the 2′-hydroxyl group migrates to the 3′-hydroxyl group during phosphoramidition of the 3′-hydroxyl group.
In addition, the use of a bulky substituent such as the TBDMS group as a 2′-hydroxyl protecting group may decrease the rate of the condensation reaction for the formation of the internucleotide bond because of steric hindrance in the vicinity of the phosphorus atom at the 3′-position, possibly resulting in cleavage or rearrangement of the internucleotide linkage during removal of the 2′-hydroxyl protecting group after oligomerization.
In order to overcome the above problems, more-efficient methods for synthesizing oligo-RNAs are now under investigation.
As a 2′-hydroxyl protecting group, the 1-(2-cyanoethoxy)ethyl (CEE) group is known to be removed together with the 3′- and 5′-protecting bissilyl group under neutral conditions capable of removing the bissilyl protecting group (Wolfgang Pfleiderer et al., Helvetica Chimica Acta, Vol. 81, 1545 (1998)).
Based on this information, Wada developed a phosphoramidite compound for producing oligo-RNAs in which the CEE group, which is capable of being removed under neutral conditions, is introduced to the 2′-hydroxy group (Takeshi Wada, Bioindustry, Vol. 21, No. 1, 17 (2004) and T. Umemoto et al., Tetrahedron Letters, Vol. 45, 9529 (2004)).
However, since the introduction of the CEE group at the 2′-hydroxyl position leads to the formation of a new asymmetric center, oligo-RNAs in which the 2′-hydroxyl groups are protected by the CEE group are a diastereoisomeric mixture. Therefore, purification and isolation of the desired oligo-RNA is complicated. In addition, since the oligo-RNAs to which the CEE group has been introduced have a methyl group on the carbon attached to the 2′-oxygen atom, some steric hindrance around the phosphorus atom attached to the 3′-hydroxyl group is expected, raising concerns about a reduction in the condensation efficiency and the condensation reaction rate.